Receptor interacting protein-2 (RIP2) kinase, which is also referred to as CARD3, RICK, CARDIAK, or RIPK2, is a TKL family serine/threonine protein kinase involved in innate immune signaling. RIP2 kinase is composed of an N-terminal kinase domain and a C-terminal caspase-recruitment domain (CARD) linked via an intermediate (IM) region ((1998) J Biol. Chem. 273, 12296-12300; (1998) Current Biology 8, 885-889; and (1998) J Biol Chem. 273, 16968-16975). The CARD domain of RIP2 kinase mediates interaction with other CARD-containing proteins, such as NODI and NOD2 ((2000) J Biol Chem. 275, 27823-27831 and (2001) EMBO reports 2, 736-742). NODI and NOD2 are cytoplasmic receptors which play a key role in innate immune surveillance. They recognize both gram positive and gram negative bacterial pathogens and are activated by specific peptidoglycan motifs, diaminopimelic acid (i.e., DAP) and muramyl dipeptide (MDP), respectively ((2007) J Immunol 178, 2380-2386).
Following activation, RIP2 kinase associates with NODI or NOD2 and appears to function principally as a molecular scaffold to bring together other kinases (TAKI, IKKα/β/γ) involved in NF-κB and mitogen-activated protein kinase activation ((2006) Nature Reviews Immunology 6, 9-20). RIP2 kinase undergoes a K63-linked polyubiquitination on lysine-209 which facilitates TAKI recruitment ((2008) EMBO Journal 27, 373-383). This post-translational modification is required for signaling as mutation of this residue prevents NOD 1/2 mediated NF-kB activation. RIP2 kinase also undergoes autophosphorylation on serine-176, and possibly other residues ((2006) Cellular Signalling 18, 2223-2229). Studies using kinase dead mutants (K47A) and non-selective small molecule inhibitors have demonstrated that RIP2 kinase activity is important for regulating the stability of RIP2 kinase expression and signaling ((2007) Biochem J 404, 179-190 and (2009) J Bioi. Chem. 284, 19183-19188).
Dysregulation of RIP2-dependent signaling has been linked to auto inflammatory diseases. Gain-of-function mutations in the NACHT-domain of NOD2 cause Blau Syndrome, early-onset sarcoidosis, a pediatric granulomateous disease characterized by uveitis, dermatitis, and arthritis ((2001) Nature Genetics 29, 19-20; (2005) Journal of Rheumatology 32, 373-375; (2005) Current Rheumatology Reports 7, 427-433; (2005) Blood 105, 1195-1197; (2005) European Journal of Human Genetics 13, 742-747; (2006) American Journal of Ophthalmology 142, 1089-1092; (2006) Arthritis & Rheumatism 54, 3337-3344; (2009) Arthritis & Rheumatism 60, 1797-1803; and (2010) Rheumatology 49, 194-196). Mutations in the LRR-domain of NOD2 have been strongly linked to susceptibility to Crohn's Disease ((2002) Am. J Hum. Genet. 70, 845-857; (2004) European Journal of Human Genetics 12, 206-212; (2008) Mucosal Immunology (2008) 1 (Suppll), 55-59. 1, S5-S9; (2008) Inflammatory Bowel Diseases 14, 295-302; (2008) Experimental Dermatology 17, 1057-1058; (2008) British Medical Bulletin 87, 17-30; (2009) Inflammatory Bowel Diseases 15, 1145-1154 and (2009) Microbes and Infection 11, 912-918). Mutations in NODI have been associated with asthma ((2005) Hum. Mol. Genet. 14, 935-941) and early-onset and extra-intestinal inflammatory bowel disease ((2005) Hum. Mol. Genet. 14, 1245-1250). Genetic and functional studies have also suggested a role for RIP2-dependent signaling in a variety of other granulomateous disorders, such as sarcoidosis ((2009) Journal of Clinical Immunology 29, 78-89 and (2006) Sarcoidosis Vasculitis and Diffuse Lung Diseases 23, 23-29) and Wegner's Granulomatosis ((2009) Diagnostic Pathology 4, 23).
A potent, selective, small molecule inhibitor of RIP2 kinase activity would block RIP2-dependent pro-inflammatory signaling and thereby provide a therapeutic benefit in auto inflammatory diseases characterized by increased and/or dysregulated RIP2 kinase activity.
It would be desirable to investigate other approaches to antagonise the RIP2 kinase.
One approach would be to develop selective RIP2 kinases down regulators or degraders that reduce RIP2 expression at either the transcript or protein level.
Several methods are available for the manipulation of protein levels, including proteolysis targeting chimeric molecules (Protacs) which contain a ligand that recognizes the target protein linked to a ligand that binds to a specific E3 ubiquitin ligase. It would be desirable to have a small molecule which can simultaneously bind RIP2 kinase and an E3 ubiquitin ligase and which promotes ubiquitination of RIP2 Kinase and leads to its degradation by the Proteosome. One suitable E3 ubiquitin ligase is the von Hippel-Lindau tumour suppressor (VHL), see for example WO2013/106643.
It would be desirable to identify further ubiquitin ligase binding molecules to incorporate into PROTAC molecules.
Inhibitors of Apoptosis (IAP) have been proposed with limited success, see for example Okuhira et al, Cell Death and Disease, 2014, 5, e1513. IAP inhibitors now known which can be of use in their own right as antitumour agents, see for example L. Bai et al./Pharmacology & Therapeutics 144 (2014) 82-95 Apoptosis is one form of programmed cell-death and is a normal cellular process used by multi-cellular organisms to eliminate damaged or unwanted cells. Apoptosis is a tightly regulated process and faulty regulation of apoptosis is implicated in many human diseases, including cancer, autoimmune diseases, inflammation, and neurogenesis (Lowe S. W and Lin 2000 Carcinogenesis 21(3), 485-495, Nicholson D. W. 2000, Nature 407 (6805) 810-816, Reed J. C. 2002 Nat Rev Drug Discovery 1(2) 111-121).
IAP inhibitors are disclosed in WO 2014031487 WO 2014047024 which describe linked dimeric compounds. WO 2014055461 describes bivalent compounds and WO 2008128171 describes IAP inhibitors all with a view to treating disorders associated with apoptosis, particularly cancer.
The present inventors have identified IAP compounds which when incorporated into PROTACs targeting RIP2 kinase are capable of promoting target degradation.